Benzothienopyridines for use as inhibitors of Eg5 kinesin

ABSTRACT

Compounds of the formula (1) 
                         
in which R1, R2, R3, R4 and X have the meanings indicated in the description, are novel effective compounds with Eg5 inhibitory, anti-proliferative and/or apoptosis inducing activity.

This application was filed under 35 U.S.C. 371 as a national stage ofPCT/EP2005/054054, filed Aug. 17, 2005.

FIELD OF APPLICATION OF THE INVENTION

The invention relates to novel classes of indolopyridine,benzofuranopyridine and benzothienopyridine derivatives, which can beused in the pharmaceutical industry for the production of pharmaceuticalcompositions.

PRIOR ART

In the document Hotha et al., Angew. Chem. 2003, 115, 2481-2484 theindolopyridine compound HR22C16 is described as inhibitor of celldivision by targeting Eg5.

EP357122 contains, inter alia, indolopyridine, benzofuranopyridine andbenzothienopyridine derivatives as cytostatic compounds.

In the International Applications WO9632003 and WO0228865 indolopyridinederivatives are described with PDE inhibitory activity.

In the International Application WO 2004/004652, inter alia,trans-10-(3-hydroxy-phenyl)-2-methyl-3a,4,9,10-tetrahydro-2,9,10a-triaza-cyclopenta[b]fluorene-1,3-dioneis described in a crystallized complex with the kinesin spindle protein(KSP).

In the US-application US 2005/0004156 indolopyridine derivatives,specifically monastroline derivatives, are described as Eg5 inhibitors.

DESCRIPTION OF THE INVENTION

It has now been found that the novel indolopyridine, benzofuranopyridineand benzothienopyridine derivatives, which are described in greaterdetails below, differ from prior art compounds by unanticipatedstructural features and have surprising and particularly advantageousproperties. Thus, for example, the compounds according to this inventioncan act as inhibitors of Eg5 kinesin. In more detail, it has beenunexpectedly found that these derivatives are potent and highlyefficacious inhibitors of cellular (hyper)proliferation and/orcell-cycle specific inducers of apoptosis in cancer cells. Therefore,unanticipatedly, these compounds can be useful for treatinghyperproliferative diseases and/or disorders responsive to the inductionof apoptosis, in particular cancer. By having a cell-cycle specific modeof action, these derivates should have a higher therapeutic indexcompared to standard chemotherapeutic drugs targeting basic cellularprocesses like DNA replication or interfering with basic cellularmolecules like DNA.

Thus, for example, the compounds according to this invention areexpected to be useful in targeted cancer therapy.

The invention thus relates in a first aspect (aspect A) to compounds offormula I.

in which

-   R1 is 1-4C-alkyl, 3-7C-cycloalkyl, 3-7C-cycloalkyl-1-4C-alkyl, or    2-7C-alkyl substituted by R11, in which-   R11 is —N(R111)R112, in which-   R111 is hydrogen, or 1-4C-alkyl,-   R112 is hydrogen, or 1-4C-alkyl,-   or R111 and R112 together and with inclusion of the nitrogen atom,    to which they are bonded, form a ring Het, in which-   Het is piperidinyl, morpholinyl, thiomorpholinyl, or pyrrolidinyl,-   R2 is hydrogen, 1-4C-alkyl, halogen, trifluoromethyl, 1-4C-alkoxy,    hydroxyl, 1-4C-alkoxy-2-4C-alkoxy, hydroxy-2-4C-alkoxy,    3-7C-cycloalkoxy, 3-7C-cycloalkyl-1-4C-alkoxy, or completely or    predominantly fluorine-substituted 1-4C-alkoxy,-   R3 is hydrogen, or 1-4C-alkoxy,    either-   X is NH, oxygen or sulphur, and-   R4 is 1-4C-alkyl,    or-   X is sulphur, and-   R4 is hydrogen,    or-   X is oxygen, and-   R4 is hydrogen,    and the salts, stereoisomers and the salts of the stereoisomers of    these compounds.

The invention further relates in a second aspect (aspect B), which is anembodiment of aspect a, to compounds of formula I,

in which

-   R1 is 1-4C-alkyl, 3-7C-cycloalkyl, 3-7C-cycloalkyl-1-4C-alkyl, or    2-7C-alkyl substituted by R11, in which-   R11 is —N(R111)R112, in which-   R111 is hydrogen, or 1-4C-alkyl,-   R112 is hydrogen, or 1-4C-alkyl,-   or R111 and R112 together and with inclusion of the nitrogen atom,    to which they are bonded, form a ring Het, in which-   Het is piperidinyl, morpholinyl, thiomorpholinyl, or pyrrolidinyl,-   R2 is hydrogen, 1-4C-alkyl, halogen, trifluoromethyl, 1-4C-alkoxy,    hydroxyl, 1-4C-alkoxy-2-4C-alkoxy, hydroxy-2-4C-alkoxy,    3-7C-cycloalkoxy, 3-7C-cycloalkyl-1-4C-alkoxy, or completely or    predominantly fluorine-substituted 1-4C-alkoxy,-   R3 is hydrogen, or 1-4C-alkoxy,-   X is NH, oxygen or sulphur,-   R4 is 1-4C-alkyl,    and the salts, stereoisomers and the salts of the stereoisomers of    these compounds.

The invention further relates in a third aspect (aspect C), which is anembodiment of aspect a, to compounds of formula I,

in which

-   R1 is 1-4C-alkyl, 3-7C-cycloalkyl, 3-7C-cycloalkyl-1-4C-alkyl, or    2-7C-alkyl substituted by R11, in which-   R11 is —N(R111)R112, in which-   R111 is hydrogen, or 1-4C-alkyl,-   R112 is hydrogen, or 1-4C-alkyl,-   or R111 and R112 together and with inclusion of the nitrogen atom,    to which they are bonded, form a ring Het, in which-   Het is piperidinyl, morpholinyl, thiomorpholinyl, or pyrrolidinyl,-   R2 is hydrogen, 1-4C-alkyl, halogen, trifluoromethyl, 1-4C-alkoxy,    hydroxyl, 1-4C-alkoxy-2-4C-alkoxy, hydroxy-2-4C-alkoxy,    3-7C-cycloalkoxy, 3-7C-cycloalkyl-1-4C-alkoxy, or completely or    predominantly fluorine-substituted 1-4C-alkoxy,-   R3 is hydrogen, or 1-4C-alkoxy,-   X is sulphur,-   R4 is hydrogen,    and the salts, stereoisomers and the salts of the stereoisomers of    these compounds.

1-4C-Alkyl is a straight-chain or branched alkyl radical having 1 to 4carbon atoms. Examples are the butyl, isobutyl, sec-butyl, tert-butyl,propyl, isopropyl, and, particularly, the ethyl and methyl radicals.

2-7C-Alkyl is a straight-chain or branched alkyl radical having 2 to 7carbon atoms. Examples are the heptyl, isoheptyl (5-methylhexyl), hexyl,isohexyl (4-methylpentyl), neohexyl (3,3-dimethylbutyl), pentyl,isopentyl (3-methylbutyl), neopentyl (2,2-dimethylpropyl), butyl,isobutyl, sec-butyl, tert-butyl, propyl, isopropyl and ethyl radicals.

3-7C-Cycloalkyl stands for cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl and cycloheptyl, of which cyclopropyl, cyclopentyl andcyclohexyl are in particular to be mentioned.

3-7C-Cycloalkyl-1-4C-alkyl stands for one of the abovementioned1-4C-alkyl radicals, which is substituted by one of the abovementioned3-7C-cycloalkyl radicals. Examples which may be mentioned are the3-7C-cycloalkylmethyl radicals, in particular the cyclopropylmethyl andthe cyclopentylmethyl radical, and the cyclohexylethyl radical.

Halogen within the meaning of the present invention is iodine or, inparticular, bromine, chlorine or fluorine.

1-4C-Alkoxy represents radicals which, in addition to the oxygen atom,contain a straight-chain or branched alkyl radical having 1 to 4 carbonatoms. Examples which may be mentioned are the butoxy, isobutoxy,sec-butoxy, tert-butoxy, propoxy, isopropoxy and preferably the ethoxyand methoxy radicals.

2-4C-Alkoxy represents radicals which, in addition to the oxygen atom,contain a straight-chain or branched alkyl radical having 2 to 4 carbonatoms. Examples which may be mentioned are the butoxy, isobutoxy,sec-butoxy, tert-butoxy, propoxy, isopropoxy and preferably the ethoxyradicals.

1-4C-Alkoxy-2-4C-alkoxy represents one of the abovementioned 2-4C-alkoxyradicals, which is substituted by one of the abovementioned 1-4C-alkoxyradicals. Examples which may be mentioned are the 2-methoxyethoxy,2-ethoxyethoxy and the 2-isopropoxyethoxy radicals.

Hydroxy-2-4C-alkoxy represents one of the abovementioned 2-4C-alkoxyradicals, which is substituted by a hydroxyl radical. Examples which maybe mentioned are the 2-hydroxyethoxy and the 3-hydroxypropoxy radicals.

3-7C-Cycloalkoxy stands for cyclopropyloxy, cyclobutyloxy,cyclopentyloxy, cyclohexyloxy or cycloheptyloxy, of whichcyclopropyloxy, cyclopentyloxy and cyclohexyloxy are in particular to bementioned.

3-7C-Cycloalkyl-1-4C-alkoxy stands for one of the abovementioned1-4C-alkoxy radicals substituted by one of the abovementioned3-7C-cycloalkyl radicals. Examples which may be mentioned are the3-7C-cycloalkylmethoxy radicals, such as cyclopropylmethoxy,cyclobutylmethoxy, cyclopentylmethoxy, cyclohexylmethoxy orcycloheptylmethoxy, of which cyclopropylmethoxy or cyclopentylmethoxyare in particular to be mentioned.

Completely or predominantly fluorine-substituted 1-4C-alkoxy is, forexample, the 2,2,3,3,3-pentafluoropropoxy, the perfluoroethoxy, the1,2,2-trifluoroethoxy and in particular the 1,1,2,2-tetrafluoroethoxy,the 2,2,2-trifluoroethoxy, the trifluoromethoxy and the difluoromethoxyradical, of which the difluoromethoxy radical is preferred.“Predominantly” in this connection means that more than half of thehydrogen atoms of the 1-4C-alkoxy groups are replaced by fluorine atoms.

Suitable salts for compounds of formula I according to thisinvention—depending on substitution—are all acid addition salts or allsalts with bases. Particular mention may be made of thepharmacologically tolerable inorganic and organic acids and basescustomarily used in pharmacy. Those suitable are, on the one hand,water-insoluble and, particularly, water-soluble acid addition saltswith acids such as, for example, hydrochloric acid, hydrobromic acid,phosphoric acid, nitric acid, sulphuric acid, acetic acid, citric acid,D-gluconic acid, benzoic acid, 2-(4-hydroxybenzoyl)benzoic acid, butyricacid, sulphosalicylic acid, maleic acid, lauric acid, malic acid,fumaric acid, succinic acid, oxalic acid, tartaric acid, embonic acid,stearic acid, toluenesulphonic acid, methanesulphonic acid or3-hydroxy-2-naphthoic acid, the acids being employed in saltpreparation—depending on whether a mono- or polybasic acid is concernedand depending on which salt is desired—in an equimolar quantitativeratio or one differing therefrom.

On the other hand, salts with bases are—depending on substitution—alsosuitable. As examples of salts with bases are mentioned the lithium,sodium, potassium, calcium, aluminium, magnesium, titanium, ammonium,meglumine or guanidinium salts, here, too, the bases being employed insalt preparation in an equimolar quantitative ratio or one differingtherefrom.

Pharmacologically intolerable salts, which can be obtained, for example,as process products during the preparation of the compounds of formula Iaccording to this invention on an industrial scale, are converted intopharmacologically tolerable salts by processes known to the personskilled in the art.

According to expert's knowledge the compounds of formula I according tothis invention as well as their salts may contain, e.g. when isolated incrystalline form, varying amounts of solvents. Included within the scopeof the invention are therefore all solvates and in particular allhydrates of the compounds of formula I according to this invention aswell as all solvates and in particular all hydrates of the salts of thecompounds of formula I according to this invention.

The substituents R2 and R3 of compounds of formula I can be attached inthe ortho, meta or para position with respect to the binding position inwhich the phenyl ring is bonded to the scaffold, whereby preference isgiven to the attachment in the meta or para position.

The compounds of formula I are chiral compounds having chiral centers inpositions 3a and 10.

The invention includes all conceivable stereoisomers, like e.g.diastereomers and enantiomers, in substantially pure form as well as inany mixing ratio, including the racemates.

Preference is given hereby to compounds of formula I, which have withrespect to the positions 3a and 10 the same configuration as shown informula I*.

If, for example, in compounds of formula I* R4 has the meaning methyl orhydrogen, then the configuration—according to the rules of Cahn, Ingoldand Prelog—is S in the 3a position and R in the 10 position.

Furthermore, compounds of the formula I also worthy to be mentioned arethose which have, with respect to the positions 3a and 10, the sameconfiguration as shown in formula I**:

If, for example, in compounds of formula I** R4 has the meaning methylor hydrogen, then the configuration—according to the rules of Cahn,Ingold and Prelog—is R in the 3a position and R in the 10 position.

Further on, compounds of the formula I also to be mentioned are thosewhich have, with respect to the positions 3a and 10, the sameconfiguration as shown in formula I*** or I****:

If, for example, in compounds of formula I*** R4 has the meaning methylor hydrogen, then the configuration—according to the rules of Cahn,Ingold and Prelog—is R in the 3a position and S in the 10 position.

If, for example, in compounds of formula I**** R4 has the meaning methylor hydrogen, then the configuration—according to the rules of Cahn,Ingold and Prelog—is S in the 3a position and S in the 10 position.

In general, enantiomerically pure compounds of this invention can beprepared according to art-known processes, such as e.g. via asymmetricsyntheses, for example by preparation and separation of appropriatediastereoisomeric compounds; by chromatographic separation on chiralseparating columns; by means of salt formation of the racemic compoundswith optically active acids or bases, subsequent resolution of the saltsand release of the desired compound from the salt; by derivatizationwith chiral auxiliary reagents, subsequent diastereomer separation andremoval of the chiral auxiliary group; or by (fractional)crystallization from a suitable solvent.

Preferably, enantiomerically pure compounds can be obtained startingfrom known enantiomerically pure starting compounds via synthesis ofdiastereomeric intermediates which can be separated by known methods(e.g. by chromatographic separation or crystallization).

In the context of this invention, hyperproliferation and analogous termsare used to describe aberrant/dysregulated cellular growth, a hallmarkof diseases like cancer. This hyperproliferation might be caused bysingle or multiple cellular/molecular alterations in respective cellsand can be, in context of a whole organism, of benign or malignantbehaviour. Inhibition of cell proliferation and analogous terms is usedto denote an ability of the compound to retard the growth of and/or killa cell contacted with that compound as compared to cells not contactedwith that compound. Most preferable this inhibition of cellproliferation is 100%, meaning that proliferation of all cells isstopped and/or cells undergo programmed cell death. In some preferredembodiments the contacted cell is a neoplastic cell. A neoplastic cellis defined as a cell with aberrant cell proliferation. A benignneoplasia is described by hyperproliferation of cells, incapable offorming an aggressive, metastasizing tumor in-vivo. In contrast, amalignant neoplasia is described by cells with different cellular andbiochemical abnormalities, e.g. capable of forming tumor metastasis. Theacquired functional abnormalities of malignant neoplastic cells (alsodefined as “hallmarks of cancer”) are replicative potential(“hyperproliferation”), self-sufficiency in growth signals,insensitivity to anti-growth signals, evasion from apoptosis, sustainedangiogenesis and tissue invasion and metastasis.

Inducer of apoptosis and analogous terms are used to identify a compoundwhich executes programmed cell death in cells contacted with thatcompound. Apoptosis is defined by complex biochemical events within thecontacted cell, such as the activation of cystein specific proteinases(“caspases”) and the fragmentation of chromatin. Induction of apoptosisin cells contacted with the compound might not necessarily be coupledwith inhibition of cell proliferation. Preferably, the inhibition ofcell proliferation and/or induction of apoptosis is specific to cellswith aberrant cell growth (hyperproliferation). Thus, compared to cellswith aberrant cell growth, normal proliferating or arrested cells areless sensitive or even insensitive to the proliferation inhibiting orapoptosis inducing activity of the compound. Finally, cytotoxic andanalogous terms is used in a more general sense to identify compoundswhich kill cells by various mechanisms, including the induction ofapoptosis/programmed cell death in a cell cycle dependent or cell-cycleindependent manner.

Cell cycle specific and analogous terms are used to identify a compoundas inducing apoptosis only in continuously proliferating cells activelypassing a specific phase of the cell cycle, but not in resting,non-dividing cells. Continuously proliferating cells are typical fordiseases like cancer and characterized by cells in all phases of thecell division cycle, namely in the G (“gap”) 1, S (“DNA synthesis”), G2and M (“mitosis”) phase.

Compounds according to aspect A of this invention more worthy to bementioned are those compounds of formula I,

in which

-   R1 is 1-2C-alkyl, cyclopropyl, cyclopropylmethyl, or 2-4C-alkyl    substituted by R11, in which-   R11 is —N(R111)R112, in which-   R111 is 1-2C-alkyl,-   R112 is 1-2C-alkyl,-   or R111 and R112 together and with inclusion of the nitrogen atom,    to which they are bonded, form a ring Het, in which-   Het is piperidinyl, morpholinyl, thiomorpholinyl, or pyrrolidinyl,-   R2 is hydrogen,-   R3 is hydrogen,    either-   X is NH, oxygen or sulphur, and

R4 is methyl,

or

-   X is sulphur, and-   R4 is hydrogen,    or-   X is oxygen, and-   R4 is hydrogen,    and the salts, stereoisomers and the salts of the stereoisomers of    these compounds.

Compounds according to aspect A of this invention in particular worthyto be mentioned are those compounds of formula I which are from formulaI* as shown below,

in which

-   R1 is methyl, ethyl, or ethyl substituted by R11, in which-   R11 is —N(R111)R112, in which-   R111 is methyl,-   R112 is methyl,-   or R111 and R112 together and with inclusion of the nitrogen atom,    to which they are bonded, form a ring Het, in which-   Het is morpholinyl,-   R2 is hydrogen,-   R3 is hydrogen,    either-   X is NH, oxygen or sulphur, and-   R4 is methyl,    or-   X is sulphur, and-   R4 is hydrogen,    or-   X is oxygen, and-   R4 is hydrogen,    and the salts of these compounds.

In one embodiment, compounds according to aspect A of this invention inmore particular worthy to be mentioned are those compounds of formula Iwhich are from formula I* as shown below,

in which

-   R1 is methyl, or ethyl substituted by R11, in which-   R11 is —N(R111)R112, in which-   R111 is methyl,-   R112 is methyl,-   R2 is hydrogen,-   R3 is hydrogen,-   X is NH, and-   R4 is methyl,    or-   X is sulphur, and-   R4 is methyl,    or-   X is oxygen, and-   R4 is methyl,    and the salts of these compounds.

In another embodiment, compounds according to aspect A of this inventionin more particular worthy to be mentioned are those compounds of formulaI which are from formula I* as shown below,

in which

-   R1 is methyl, or ethyl substituted by R11, in which-   R11 is —N(R111)R112, in which-   R111 is methyl,-   R112 is methyl,-   R2 is hydrogen,-   R3 is hydrogen,    either-   X is sulphur, and-   R4 is hydrogen,    or-   X is oxygen, and-   R4 is hydrogen,    and the salts of these compounds.

In one embodiment, compounds according to aspect A of this invention tobe emphasized are those compounds of formula I which are from formula I*as shown below,

in which

-   R1 is methyl,-   R2 is hydrogen,-   R3 is hydrogen,    either-   X is NH, and-   R4 is methyl,    or-   X is sulphur, and-   R4 is methyl,    or-   X is oxygen, and-   R4 is methyl,    and the salts of these compounds.

In another embodiment, compounds according to aspect A of this inventionto be emphasized are those compounds of formula I which are from formulaI* as shown below,

in which

-   R1 is methyl,-   R2 is hydrogen,-   R3 is hydrogen,    either-   X is sulphur, and-   R4 is hydrogen,    or-   X is oxygen, and-   R4 is hydrogen,    and the salts of these compounds.

Compounds according to aspect B of this invention more worthy to bementioned are those compounds of formula I,

in which

-   R1 is 1-4C-alkyl, 3-5C-cycloalkyl, 3-5C-cycloalkyl-1-4C-alkyl, or    2-7C-alkyl substituted by R11, in which-   R11 is —N(R111)R112, in which-   R111 is hydrogen,-   R112 is hydrogen,-   R2 is hydrogen,-   R3 is hydrogen,-   X is NH, oxygen or sulphur,-   R4 is methyl,    and the salts, stereoisomers and the salts of the stereoisomers of    these compounds.

Compounds according to aspect B of this invention in particular worthyto be mentioned are those compounds of formula I,

in which

-   R1 is 1-2C-alkyl, cyclopropyl, cyclopropylmethyl, or 2-amino-ethyl,-   R2 is hydrogen,-   R3 is hydrogen,-   X is NH, oxygen or sulphur,-   R4 is methyl,    and the salts, stereoisomers and the salts of the stereoisomers of    these compounds.

Compounds according to aspect B of this invention in more particularworthy to be mentioned are those compounds of formula I,

in which

-   R1 is methyl, ethyl, or cyclopropyl,-   R2 is hydrogen,-   R3 is hydrogen,-   X is NH, oxygen or sulphur,-   R4 is methyl,    and the salts, stereoisomers and the salts of the stereoisomers of    these compounds.

Compounds according to aspect B of this invention to be emphasized arethose compounds of formula I,

in which

-   R1 is methyl,-   R2 is hydrogen,-   R3 is hydrogen,-   X is NH, oxygen or sulphur,-   R4 is methyl,    and the salts, stereoisomers and the salts of the stereoisomers of    these compounds.

Compounds according to aspect C of this invention more worthy to bementioned are those compounds of formula I,

in which

-   R1 is 1-4C-alkyl, 3-5C-cycloalkyl, 3-5C-cycloalkyl-1-4C-alkyl, or    2-7C-alkyl substituted by R11, in which-   R11 is —N(R111)R112, in which-   R111 is hydrogen,-   R112 is hydrogen,-   R2 is hydrogen,-   R3 is hydrogen,-   X is sulphur,-   R4 is hydrogen,    and the salts, stereoisomers and the salts of the stereoisomers of    these compounds.

Compounds according to aspect C of this invention in particular worthyto be mentioned are those compounds of formula I,

in which

-   R1 is 1-2C-alkyl, cyclopropyl, cyclopropylmethyl, or 2-amino-ethyl,-   R2 is hydrogen,-   R3 is hydrogen,-   X is sulphur,-   R4 is hydrogen,    and the salts, stereoisomers and the salts of the stereoisomers of    these compounds.

Compounds according to aspect C of this invention in more particularworthy to be mentioned are those compounds of formula I,

in which

-   R1 is methyl, ethyl, or cyclopropyl,-   R2 is hydrogen,-   R3 is hydrogen,-   X is sulphur,-   R4 is hydrogen,    and the salts, stereoisomers and the salts of the stereoisomers of    these compounds.

A special interest in the compounds according to this invention refersto those compounds of formula I which are included—within the scope ofthis invention—by one or, when possible, by more of the followingspecial embodiments:

A special embodiment (embodiment 1) of the compounds of formula Iaccording to this invention refers to those compounds of formula I, inwhich

-   R1 is methyl.

Another special embodiment (embodiment 2) of the compounds of formula Iaccording to this invention refers to those compounds of formula I, inwhich

-   R2 is hydrogen.

Another special embodiment (embodiment 3) of the compounds of formula Iaccording to this invention refers to those compounds of formula I, inwhich

-   R3 is hydrogen.

Another special embodiment (embodiment 4) of the compounds of formula Iaccording to this invention refers to those compounds of formula I, inwhich

-   R2 and R3 are both hydrogen.

Another special embodiment (embodiment 5) of the compounds of formula Iaccording to this invention refers to those compounds which are fromformula I* as shown above.

Another special embodiment (embodiment 6) of the compounds of formula Iaccording to this invention refers to those compounds of formula I, inwhich

-   R4 is methyl.

Another special embodiment (embodiment 7) of the compounds of formula Iaccording to this invention refers to those compounds of formula I, inwhich

-   X is NH, and R4 is methyl.

Another special embodiment (embodiment 8) of the compounds of formula Iaccording to this invention refers to those compounds of formula I, inwhich

-   X is oxygen, and R4 is methyl.

Another special embodiment (embodiment 9) of the compounds of formula Iaccording to this invention refers to those compounds of formula I, inwhich

-   X is sulphur, and R4 is methyl.

Another special embodiment (embodiment 10) of the compounds of formula Iaccording to this invention refers to those compounds of formula I, inwhich

-   R4 is hydrogen.

Another special embodiment (embodiment 11) of the compounds of formula Iaccording to this invention refers to those compounds of formula I, inwhich

-   X is sulphur, and R4 is hydrogen.

Another special embodiment (embodiment 12) of the compounds of formula Iaccording to this invention refers to those compounds of formula I, inwhich

-   X is oxygen, and R4 is hydrogen.

Another special embodiment (embodiment 13) of the compounds of formula Iaccording to this invention refers to those compounds of formula I, inwhich

-   R1 is 2-(N,N-dimethylamino)-ethyl.

A particular special embodiment (embodiment 14) of the compounds offormula I according to this invention refers to those compounds offormula I*, in which

-   X is NH, R1 is 2-(N,N-dimethylamino)-ethyl, and R4 is methyl.

Another particular special embodiment (embodiment 15) of the compoundsof formula I according to this invention refers to those compounds offormula I*, in which

-   X is sulphur, R1 is 2-(N,N-dimethylamino)-ethyl, and R4 is methyl.

Another particular special embodiment (embodiment 16) of the compoundsof formula I according to this invention refers to those compounds offormula I*, in which

-   X is sulphur, R1 is 2-(N,N-dimethylamino)-ethyl, and R4 is hydrogen.

A further particular special embodiment (embodiment 13) of the compoundsof formula I according to this invention refers to those compounds offormula I*, in which

-   X is NH, R1 is methyl, and R4 is methyl.

Another further particular special embodiment (embodiment 14) of thecompounds of formula I according to this invention refers to thosecompounds of formula I*, in which

-   X is sulphur, R1 is methyl, and R4 is methyl.

Another further particular special embodiment (embodiment 15) of thecompounds of formula I according to this invention refers to thosecompounds of formula I*, in which

-   X is sulphur, R1 is methyl, and R4 is hydrogen.

As exemplary compounds according to this invention the followingcompounds of formula I* in which R2 and R3 are both hydrogen, can bementioned by means of the substituent meanings for R1, R4 and X in thefollowing Table 1.

TABLE 1 No. R1 R4 X 1.1 —CH₃ —CH₃ NH 1.2 —CH₃ —CH₃ S 1.3 —CH₂CH₂—N(CH₃)₂—CH₃ NH 1.4 —CH₂CH₂—N(CH₃)₂ —CH₃ S 1.5 —CH₃ H S 1.6 —CH₂CH₂—N(CH₃)₂ H S

The compounds according to the invention can be prepared e.g. asdescribed exemplarily as follows and according to the followingspecified reaction steps, or, particularly, in a manner as described byway of example in the following examples, or analogously or similarlythereto according to preparation procedures or synthesis strategiesknown to the person skilled in the art.

As shown in the synthesis route outlined in scheme 1 below, compounds offormula IV, in which X and R4 have the meanings given above, arecondensed and cyclized in a Pictet-Spengler reaction with benzaldehydesof formula III, in which R2 and R3 have the meanings mentioned above, togive the corresponding compounds of formulae IIa and/or IIb mostly as amixture. Said Pictet-Spengler reaction can be carried out as it is knownto the skilled person or as described in the following examples,advantageously in the presence of a suitable acid as a catalyst orpromotor (e.g. trifluoroacetic acid) in a suitable solvent, for exampletoluene, at elevated temperature.

Compounds of formula IV, in which X and R4 have the meanings givenabove, are known or can be obtained in a known manner, e.g. byesterification of corresponding compounds of formula V which are knownor obtainable in a known manner. Thus, e.g.2-amino-3-(1H-indol-3-yl)-2-methyl-propionic acid methyl ester iscommercially available. Further on,(R)-2-amino-3-(benzothiophen-3-yl)-propionic acid methyl ester isobtained from D-thiotryptophan by esterification reaction. Saidesterification reaction can be carried out in a manner habitual per seto the skilled person, e.g. via an appropriate corresponding activatedform of the acid, such as, for example, the corresponding acidchloride—obtainable with the aid of thionyl chloride or the like—whichis reacted with the corresponding alcohol, preferably methanol.D-Thiotryptophan is known or can be obtained in a known manner.

Moreover, compounds of formula IV or V, in which R4 is 1-4C-alkyl,particularly methyl can be obtained by asymmetric synthesis. Thus, e.g.enantiomerically pure compounds of formula IV or V, in which R4 is1-4C-alkyl, particularly methyl, can be obtained from the correspondingcompounds of the formula IV or V, respectively, in which R4 is hydrogen,by art-known stereoselective alkylation reaction, such as e.g.(S)-α-methyltryptophan can be obtained from L-tryptophane as describedin J. Org. Chem. 1995, 60, 5719-5720.

Compounds of formula III are known or can be obtained in a known manner.

The compounds of formula IV can be employed in the abovementionedPictet-Spengler reaction as racemate or enantiomerically pure compounds.Depending thereon, the mixture obtained can contain the compounds offormulae IIa and IIb as diastereomers or as diastereomeric racemate.Said mixture can be optionally separated in a manner habitual per se tothe skilled person, such as e.g. diastereomeric compounds of formulaeIIa and IIb can be separated by column chromatography. If appropriate,said mixture can be also used in the next step without furtherseparation of the diastereoisomers. Then, separation of diastereomerscan be carried out subsequently to one of the following steps.

When the compounds of formula IV, in which R4 is 1-4C-alkyl,particularly methyl, are employed as racemic mixture in theabovementioned Pictet-Spengler reaction, the racemate comprising theenantiomeric compounds of formulae IIa′ and IIb′, in each of which R4 R4is 1-4C-alkyl, particularly methyl, can be obtained preferentially or inexcess from said reaction.

Starting from the appropriate pure enantiomers of the compounds offormula IV, in which R4 is 1-4C-alkyl, particularly methyl,corresponding compounds of either formula IIa′ or formula IIb′(depending from the configuration of the starting compound of formulaIV) can be obtained preferentially. Thus, e.g. when(S)-α-methyltryptophan methyl ester [i.e.(S)-2-amino-3-(1H-indol-3-yl)-2-methyl-propionic acid methyl ester] isemployed in the abovementioned Pictet-Spengler reaction, correspondingcompounds of formula IIa′, in which X is NH and R4 is methyl, areobtained preferentially.

When the compounds of formula IV, in which R4 is hydrogen, are employedas racemic mixture in the abovementioned Pictet-Spengler reaction, theracemate comprising the enantiomeric compounds of formulae IIa″ andIIb″, in each of which R4 is hydrogen, can be obtained preferentially orin excess from said reaction.

Starting from the appropriate pure enantiomers of the compounds offormula IV, in which R4 is hydrogen, corresponding compounds of eitherformula IIa″ or formula IIb″ (depending from the configuration of thestarting compound of formula IV) can be obtained preferentially. Thus,e.g. when (R)-2-amino-3-(benzothiophen-3-yl)-propionic acid methyl esteris employed in the abovementioned Pictet-Spengler reaction,corresponding compounds of formula IIa″, in which X is S and R4 ishydrogen, are obtained preferentially.

Compounds of formula IIa′ or IIb′, in each of which R4 is 1-4C-alkyl,particularly methyl, e.g. in enantiomerically pure form or as racemicmixture or with corresponding diastereomers co-generated in thePictet-Spengler reaction above, can be reacted with isocyanates offormula R1-N═C═O or with corresponding activated carbamic acid esters,such as, for example, N-hydroxysuccinimid-activated urethanes, like e.g.H₃C—NH—C(O)—OR, in which R is 1N-succinimidyl, in a Hydantoin synthesisas shown in reaction scheme 2 to give the corresponding desiredhydantoins of formula I* (from compounds of formula IIa′) or I*** (fromcompounds of formula IIb′), in each of which R4 is 1-4C-alkyl,particularly methyl. Said Hydantoin synthesis can be performed in anart-known manner or as described in the following examples, e.g. in thepresence of microwaves. When the compounds of formulae I* and I*** areobtained as racemic mixture, the enantiomerically pure compounds may beaccessible by art-known separation techniques, such as e.g. thosedescribed above or as specified in the following examples.

Isocyanates of formula R1-N═C═O, in which R1 has the meanings givenabove, are known or can be obtained according to known procedures. Thus,e.g. compounds of formula R1-N═C═O, in which R1 is 2-7C-alkylsubstituted by —N(R111)R112, can be obtained from compounds of formulaR1-N═C═O, in which R1 is 2-7C-alkyl substituted by a suitable leavinggroup, such as e.g. bromine, by nucleophilic substitution reaction withcorresponding amines of formula HN(R111)R112 in a manner habitual per seto the skilled person or similarly as described by way of example in thefollowing example.

Compounds of formula IIa″ or IIb″, in each of which R4 is hydrogen, e.g.in enantiomerically pure form or as racemic mixture or withcorresponding diastereomers co-generated in the Pictet-Spengler reactionabove, can be reacted with isocyanates of formula R1-N═C═O or withcorresponding activated carbamic acid esters, such as, for example,N-hydroxysuccinimid-activated urethanes, like e.g. H₃C—NH—C(O)—OR, inwhich R is 1N-succinimidyl, in a Hydantoin synthesis as shown inreaction scheme 3 to give the corresponding desired hydantoins offormula I** (from compounds of formula IIa″) or I**** (from compounds offormula IIb″), in each of which R4 is hydrogen. Said Hydantoin synthesiscan be performed in an art-known manner or as described in the followingexamples, e.g. in the presence of microwaves.

Optionally, the configuration of the chiral carbon atom 3a of compoundsof formula I, in which R4 is hydrogen, can be epimerized viadeprotonation/reprotonation with the aid of a suitable base such as e.g.potassium carbonate in a suitable solvent such as e.g. acetonitrile.

Thus e.g. as shown in reaction scheme 4, compounds of formula I**, inwhich X, R1, R2 and R3 have the meanings indicated above and R4 ishydrogen, can be converted into corresponding compounds of formula I*.

Likewise, depending on the reaction conditions used, at least partialepimerization of the configuration of the CH atom adjacent to thecarbonyl group can take place in the aforementioned Hydantoin synthesis.

When the compounds of formulae I* and I*** are obtained as racemicmixture, the corresponding enantiomerically pure compounds may beaccessible by art-known separation techniques, such as e.g. thosedescribed above.

Optionally, compounds of the formula I can be converted into theirsalts, or, optionally, salts of the compounds of the formula I can beconverted into the free compounds.

It is moreover known to the person skilled in the art that if there area number of reactive centers on a starting or intermediate compound itmay be necessary to block one or more reactive centers temporarily byprotective groups in order to allow a reaction to proceed specificallyat the desired reaction center. A detailed description for the use of alarge number of proven protective groups is found, for example, in“Protective Groups in Organic Synthesis” by T. Greene and P. Wuts (JohnWiley & Sons, Inc. 1999, 3^(rd) Ed.) or in “Protecting Groups (ThiemeFoundations Organic Chemistry Series N Group” by P. Kocienski (ThiemeMedical Publishers, 2000).

The substances according to the invention are isolated and purified in amanner known per se, for example by distilling off the solvent underreduced pressure and recrystallizing the residue obtained from asuitable solvent or subjecting it to one of the customary purificationmethods, such as, for example, column chromatography on a suitablesupport material.

Salts can be obtained by dissolving the free compound in a suitablesolvent (e.g. a ketone, such as acetone, methyl ethyl ketone or methylisobutyl ketone, an ether, such as diethyl ether, tetrahydrofuran ordioxane, a chlorinated hydrocarbon, such as methylene chloride orchloroform, or a low-molecular-weight aliphatic alcohol, such as ethanolor isopropanol) which contains the desired acid or base, or to which thedesired acid or base is then added. The salts can be obtained byfiltering, reprecipitating, precipitating with a nonsolvent for theaddition salt or by evaporating the solvent. Salts obtained can beconverted into the free compounds, which can in turn be converted intosalts, by alkalization or by acidification. In this manner,pharmacologically unacceptable salts can be converted intopharmacologically acceptable salts.

Suitably, the conversions mentioned in this invention can be carried outanalogously or similarly to methods which are familiar per se to theperson skilled in the art.

The person skilled in the art knows on the basis of his/her knowledgeand on the basis of those synthesis routes, which are shown anddescribed within the description of this invention, how to find otherpossible synthesis routes for compounds according to this invention. Allthese other possible synthesis routes are also part of this invention.

Having described the invention in detail, the scope of the presentinvention is not limited only to those described characteristics orembodiments. As will be apparent to persons skilled in the art,modifications, analogies, variations, derivations, homologisations andadaptations to the described invention can be made on the base ofart-known knowledge and/or, particularly, on the base of the disclosure(e.g. the explicite, implicite or inherent disclosure) of the presentinvention without departing from the spirit and scope of this inventionas defined by the appended claims.

The following examples serve to illustrate the invention further withoutrestricting it. Likewise, further compounds according to this invention,whose preparation is not explicitly described, can be prepared in ananalogous or similar manner or in a manner familiar per se to the personskilled in the art using customary process techniques.

Any or all of the compounds of formula I according to this inventionwhich are mentioned as final compounds in the following examples, andparticularly those enantiomers thereof having the formula I*, as well asthe salts of these compounds and enantiomers, are a preferred subject ofthe present invention.

In the examples, m.p. stands for melting point, h for hour(s), min forminutes, conc. for concentrated, calc. for calculated, fnd. for found, Mfor molecular ion in mass spectrometry, and other abbreviations havetheir meanings customary per se to the skilled person.

According to common practice in stereochemistry, the symbols RS and SRare used to denote the specific configuration of each of the indicatedchiral centers of a racemate. In more detail, for example, the term“(3aSR,10RS)” stands for a racemate comprising the one enantiomer havingthe configuration (3aS,10R) and the other enantiomer having theconfiguration (3aR,10S).

EXAMPLES Final Compounds 1.(±)-(3aSR,10RS)-10-(3-Hydroxy-phenyl)-2,3a-dimethyl-3a,4,9,10-tetrahydro-2,9,10a-triaza-cyclopenty[b]fluorene-1,3-dione

To a suspension of 250 mg (740 μmol)(1RS,3SR)-1-(3-hydroxy-phenyl)-3-methyl-2,3,4,9-tetrahydro-1H-β-carboline-3-carboxylicacid methyl ester (compound A1) in 4 ml acetonitrile and 1 ml water areadded 511 mg (3 mmol) N-succinimidyl-N-methylcarbamate. The mixture isheated to 150° C. for 5 min using a microwave reactor. The solvents areremoved under reduced pressure. The residue is dissolved in ethylacetate and the organic layer is washed with water. The solution isdried with magnesium sulfate and the solvent is removed under reducedpressure. After column chromatography (toluene, ethyl acetate 4:1), 68mg (25%) of the title compound are obtained as a white solid (m.p.:299-305° C.; m/z (MH⁺)=362.2).

2.(3aS,10R)-10-(3-Hydroxy-phenyl)-2,3a-dimethyl-3a,4,9,10-tetrahydro-2,9,10a-triaza-cyclopenty[b]fluorene-1,3-dione

Starting from the corresponding racemate (Example 1) the title compoundas well as(3aR,10S)-10-(3-hydroxy-phenyl)-2,3a-dimethyl-3a,4,9,10-tetrahydro-2,9,10a-triaza-cyclopenty[b]fluorene-1,3-dionecan be obtained by chromatographical separation using the followingconditions: Column: 250×20 mm Chiralpak OD-I 20 μm; Mobile phase: 80/20CO2/MeOH (sample in DMSO/MeOH 50/50); Flow rate: 60 ml/min; Detection:UV 295 nm; Temperature: 25° C.; Outlet pressure: 150 bar.

Using these conditions, the title compound is obtained as second eluatedcompound.

3.(3aS,10R)-10-(3-Hydroxy-phenyl)-2-methyl-4,10-dihydro-3aH-9-thia-2,10-diaza-cyclopenta[b]fluorene-1,3-dione

Crude(3aR,10R)-10-(3-hydroxy-phenyl)-2-methyl-4,10-dihydro-3aH-9-thia-2,10-diaza-cyclo-penta[b]-fluorene-1,3-dione(compound A3) obtainable as described below is dissolved in 10 ml ofacetonitrile. 815 mg (5.90 mmol) of potassium carbonate are added andthe suspension is heated to reflux for 2 h. The solvent is removed underreduced pressure. Water and ethyl acetate are added and the aqueouslayer is washed with ethyl acetate. The combined organic phases arewashed with brine and dried with magnesium sulfate. The solvent isremoved under reduced pressure. After the addition of diisopropyl etherto the residue, 48 mg (22%) of the title compound can be obtained aswhite crystals. (m.p. 213.3° C.-216.4° C., m/z (MH⁺)=365.1).

4.(3aS,10R)-2-Butyl-10-(3-hydroxy-phenyl)-4,10-dihydro-3aH-9-thia-2,10a-diaza-cyclopenta[b]fluorene-1,3-dione

The title compound is prepared similarly as described to attain toExample 3 using butyl isocyanate instead ofN-succinimidyl-N-methylcarbamate.

C, 23; H, 22; N, 2; O, 3; S (calc.: 406.51). Fnd.: m/z (MH⁺)=362.2.

5.(3aSR,10RS)-10-(3-Hydroxy-phenyl)-2,3a-dimethyl-4,10-dihydro-3aH-9-thia-2,10a-diaza-cyclopenta[b]fluorene-1,3-dione

The title compound is prepared similarly as described to attain toExample 1 using compound A5 as starting material

C, 21; H, 18; N, 2; O, 3; S (calc.: 378.45). Fnd.: m/z (MH⁺)=379.0.

6.(3aS,10R)-10-(3-Hydroxy-phenyl)-2,3a-dimethyl-4,10-dihydro-3aH-9-thia-2,10a-diaza-cyclopenta[b]fluorene-1,3-dione

Starting from the corresponding racemate (Example 5) the title compoundmay be obtained by chromatographical separation.

7.(3aSR,10RS)-2-(2-Dimethylamino-ethyl)-10-(3-hydroxy-phenyl)-3a-methyl-3a,4,9,10-tetrahydro-2,9,10a-triaza-cyclopenty[b]fluorene-1,3-dione

The title compound may be prepared using similar procedures to thosedescribed in Example 1, but with choice of compound A6 as startingmaterial.

8.(3aS,10R)-2-(2-Dimethylamino-ethyl)-10-(3-hydroxy-phenyl)-3a-methyl-3a,4,9,10-tetrahydro-2,9,10a-triaza-cyclopenty[b]fluorene-1,3-dione

Starting from the corresponding racemate (Example 7) the title compoundmay be obtained by chromatographical separation.

9.(3aS,10R)-2-(2-Dimethylamino-ethyl)-10-(3-hydroxy-phenyl)-4,10-dihydro-3aH-9-thia-2,10-diaza-cyclopenta[b]fluorene-1,3-dione

The title compound may be prepared using similar procedures to thosedescribed to attain to Example 3, but with choice of compound A6 asstarting material.

10.(3aSR,10RS)-2-(2-Dimethylamino-ethyl)-10-(3-hydroxy-phenyl)-3a-methyl-4,10-dihydro-3aH-9-thia-2,10a-diaza-cyclopenta[b]fluorene-1,3-dione

The title compound may be prepared using similar procedures to thosedescribed to attain to Example 5, but with choice of compound A6 asstarting material.

11.(3aS,10R)-2-(2-Dimethylamino-ethyl)-10-(3-hydroxy-phenyl)-3a-methyl-4,10-dihydro-3aH-9-thia-2,10a-diaza-cyclopenta[b]fluorene-1,3-dione

Starting from the corresponding racemate (Example 10) the title compoundmay be obtained by chromatographical separation.

Starting Compounds

A1.(±)-(1RS,3SR)-1-(3-Hydroxy-phenyl)-3-methyl-2,3,4,9-tetrahydro-1H-β-carboline-3-carboxylicacid methyl ester

1.00 g (4.30 mmol) of the commercially available2-amino-3-(1H-indol-3-yl)-2-methyl-propionic acid methyl ester arereacted with 600 mg (4.73 mmol) 3-hydroxy-benzaldehyde in the presenceof trifluoroacetic acid according to the Pictet-Spengler reaction. Theresulting mixture of diastereomers is separated by column chromatography(toluene, ethyl acetate 4:1). 1.17 g (68%) of the title compound and 320mg (14%) of(1SR,3SR)-1-(3-hydroxy-phenyl)-3-methyl-2,3,4,9-tetrahydro-1H-β-carboline-3-carboxylicacid methyl ester (compound A2) are obtained as white solids.

Compound A1: m.p.: 227.5° C.; m/z (MH⁺): 337.0

Compound A2: m.p.: 221.5° C.; m/z (MH⁺): 336.9

A3.(3aR,10R)-10-(3-Hydroxy-phenyl)-2-methyl-4,10-dihydro-3aH-9-thia-2,10-diaza-cyclopenta[b]fluorene-1,3-dione

405 mg (2.35 mmol) of N-succinimidyl-N-methyl carbamate are added to asolution of 200 mg (590 μmol) of(1R,3R)-1-(3-hydroxy-phenyl)-1,2,3,4-tetrahydro-benzo[4,5]thieno[2,3-c]pyridine-3-carboxylicacid methyl ester (compound A4) in 4 ml of acetone. The mixture isheated to 150° C. for 5 min. using a microwave reactor. The solvent isremoved under reduced pressure.

The crude product obtained can be used as starting material to obtaincompound 3.

A4.(1R,3R)-1-(3-Hydroxy-phenyl)-1,2,3,4-tetrahydro-benzo[4,5]thieno[2,3-c]pyridine-3-carboxylicacid methyl ester

1.45 g (11.9 mmol) of 3-hydroxy benzaldehyde and 4.2 ml (54.6 mmol) oftrifluoro acetic acid are added to a solution of 2.04 g (8.7 mmol) of2-amino-3-benzo[b]thiophen-3-yl-propionic acid methyl ester (compoundB1) in 20 ml of toluene. The solution is stirred at 40° C. over night.The mixture is washed with a saturated solution of sodium hydrogencarbonate. The aqueous layer is washed with ethyl acetate and thecombined organic layers are washed with 5 M hydrochloric acid and driedwith magnesium sulfate. 3.61 g of a brown oil are obtained as the crudeproduct.

The title compound is separated from byproducts and from the resultingdiastereomer by column chromatography (silica gel, toluene/ethyl acetate9:1). 960 mg (32%) are obtained as a white solid (m.p. 282.9° C.-285.3°C., m/z (MH⁺)=381.1).

A5.(±)-trans-1-(3-Hydroxyphenyl)-5-methyl-5-methoxycarbonyl-1,2,3,4-tetrahydrobenzo[4,5]-thieno[2,3]pyridine

To a solution of 1.19 g of compound B2 in toluene 3-hydroxybenzaldehyde(0.80 g) and trifluoroacetic acid (2.30 mL) are added, and the mixtureis stirred at 40° C. After 2 days the mixture is made alkaline withaqueous NaHCO₃ and is extracted with ethyl acetate (3×50 mL). Thecombined organic layer is washed with water, dried and concentrated.Column chromatography (toluene-acetone, 9:1) of the residue gives thetwo diastereomers of which the title compound is the faster migratingone (m.p. 214-215° C. (from ethyl acetate—light petroleum)).

A6. (2-Isocyanato-ethyl)-dimethyl-amine

2.50 g 2-bromoethylisocyanate are dissolved in 20 ml dichloromethane. Aweak flow of dimethylamine is bubbled through the solution for 3 hours.

The solvent is removed at reduced pressure. 1.9 g of the title compoundare obtained as a colourless oil, which can be used without furtherpurification (m/z (M⁺)=114.1.

B1. 2-Amino-3-benzo[b]thiophen-3-yl-propionic acid methyl ester

A suspension of 5.00 g (22.6 mmol) H-β-(3-benzothienyl)-D-Ala-OH in 70ml of methanol is cooled to 0° C. 8.3 ml (113 mmol) of thienyl chlorideare added dropwise (the temperature rises up to 10° C. during theaddition). The solution is stirred at 0° C. for 1 hour and at roomtemperature over night. The solvent is removed under reduced pressureand the residue is washed with a saturated solution of sodium hydrogencarbonate. The aqueous phase is extracted with ethyl acetate. Thecombined organic layers are washed with brine and dried with magnesiumsulfate. The solvent is removed under reduced pressure. 5.37 g (quant.)of the title compound are obtained as a pale yellow oil (m/z(MH⁺)=236.0).

B2. (±)-Methyl 2-amino-3-benzo[b]thiophene-3-yl-2-methyl-propionate

To a solution of potassium t-butoxide (4.82 g) in dry tetrahydrofuran(60 mL) cooled to −30° C. a solution of 6.85 g of(±)-2-(benzylidene-amino)-propionic acid methyl ester (compound C2) intetrahydrofuran (20 mL) is added dropwise (compound C2 is prepared asdescribed in J. W. Tilley, P. Levitan, R. W. Kierstead, J. Heterocycl.Chem., 16, 333 (1979) and P. Bey, J.-P. Vevert, V. Van Dorsselaer, M.Kolb, J. Org. Chem., 44, 2732 (1979)). The mixture is stirred at −30° C.for 30 minutes, then a solution of 7.85 g of compound C1 intetrahydrofuran (20 mL) is added, and the mixture is stirred at the sametemperature. When TLC (toluene-acetone, 95:5) indicates thedisappearance of the starting material (cca 6 h), the mixture is dilutedwith dichloromethane, washed with water, dried and the solvent isevaporated. The residue is dissolved in dichloromethane (30 mL), cooledin an ice bath, and an ethereal solution of HCl is added to pH cca 1.The mixture is stirred for 1 h and the solvent is removed at reducedpressure. The residue is taken up in methanol, and is made alkaline withAmberlite IR 400 [OH⁻] resin. The resin is filtered off, washed withmethanol, and the filtrate is concentrated. Column chromatography of theresidue (toluene-acetone, 4:1) affords the title compound (4.28 g, 48%)as a syrup, which crystallizes on standing. M.p. 44-45° C. (from ethylacetate—light petroleum).

C1. 3-Chloromethyl-benzo[b]thiophene

The title compound is prepared following S. Avakian, J. Moss and G. J.Martin, J. Am. Chem. Soc., 70, 3075 (1948) and F. F. Blicke, D. G.Sheets, J. Am. Chem. Soc., 70, 3768 (1948).

A rapid stream of hydrogen chloride gas is bubbled through an intenselystirred mixture of benzothiophene (40.26 g), 36% aqueous formaldehyde(80 mL) and concentrated hydrochloric acid (20 mL) cooled in an icebath. After 20 minutes the mixture is diluted with ice-water, and isextracted with dichloromethane (3×200 mL). The combined organic phase iswashed with aqueous NaHCO₃, dried and the solvent is evaporated. Vacuumdistillation of the residue affords the title compound (28.9 g, 52%) asthe main fraction boiling at 128-132° C. (7.1 mbar), which crystallizeson standing.

Commercial Utility

The compounds according to the present invention have valuablepharmacological properties which can make them commercially applicable.Thus, for example, the compounds according to this invention can act asinhibitors of the mitotic kinesin Eg5 and these compounds are expectedto be commercially applicable in the therapy of diseases responsive tothe inhibition of this kinesin, such as e.g. those diseases mentionedbelow. Also, for example, the compounds according to this invention candisplay cell-cycle dependent, anti-proliferative and/or apoptosisinducing activity.

The mitotic kinesin Eg5 is an enzyme essential for the assembly andfunction of the bipolar mitotic spindle. Eg5 plays essential rolesduring all phases of mitosis. Drugs that perturb mitosis have provenclinically effective in the treatment of many cancers. Despite thediverse array of essential spindle proteins that could be exploited astargets for the discovery of novel cancer therapies, allspindle-targeted therapeutics in clinical use today act on only oneprotein, tubulin. Surprisingly, kinesin Eg5 expression is most abundantin proliferating human tissues, whereas it is absent from mostpostmitotic cells, such as e.g. human central nervous system neurons,consistent with an exclusive or almost confined role for Eg5 in cellproliferation. In contrary to drugs that directly interfere withmicrotubule dynamic instability, Eg5 kinesin inhibitors are expected notto disrupt microtubule-based cellular processes, e.g. neuronal vesicletransport, that are unrelated to proliferation. During mitosis, Eg5 isessentially involved in organizing microtubules into a bipolar structurethat forms the mitotic spindle. Experimental perturbation of Eg5function causes a characteristic malformation or dysfunction of themitotic spindle, frequently resulting in cell cycle arrest and celldeath.

The compounds according to this invention can be used to modulatemitotic spindle formation, thus causing prolonged cell cycle arrest inmitosis, which is frequently followed by apoptosis. By “modulate” hereinis meant altering mitotic spindle formation, including increasing anddecreasing spindle formation. By “mitotic spindle formation” herein ismeant organization of microtubules into bipolar structures by mitotickinesins. By “dysfunction of the mitotic spindle” herein is meantmitotic arrest and monopolar spindle formation. “Malformation of themitotic spindle” encompasses the splaying of mitotic spindle poles, orotherwise causing morphological perturbation of the mitotic spindle.

Further on, these compounds can be useful in the treatment of benign ormalignant neoplasia. A “neoplasia” is defined by cells displayingaberrant cell proliferation and/or survival and/or a block indifferentiation. A “benign neoplasia” is described by hyperproliferationof cells, incapable of forming an aggressive, metastasizing tumorin-vivo. In contrast, a “malignant neoplasia” is described by cells withmultiple cellular and biochemical abnormalities, capable of forming asystemic disease, for example forming tumor metastasis in distantorgans.

Various diseases are caused by limitless replicative potential andaberrant cell proliferation (“hyperproliferation”) as well as evasionfrom apoptosis. These diseases include benign hypoplasia like that ofthe prostate (“BPH”) or colon epithelium. Most importantly thesediseases include malignant neoplasia commonly described as cancer andcharacterized by tumor cells finally metastasizing into distinct organsor tissues. Malignant neoplasia includes solid and hematological tumors.Solid tumors are exemplified by tumors of the breast, bladder, bone,brain, central and peripheral nervous system, colon, endocrine glands(eg thyroid and adrenal cortex), esophagus, endometrium, germ cells,head and neck, kidney, liver, lung, larynx and hypopharynx,mesothelioma, sarcoma, ovary, pancreas, prostate, rectum, renal, smallintestine, soft tissue, testis, stomach, skin, ureter, vagina and vulva.Malignant neoplasia include inherited cancers exemplified byretinomblastoma and Wilms tumor. In addition, malignant neoplasiainclude primary tumors in said organs and corresponding secondary tumorsin distant organs (“tumor metastases”). Hematological tumors areexemplified by aggressive and indolent forms of leukemia and lymphoma,namely non-Hodgkins disease, chronic and acute myeloid leukemia(CML/AML), acute lymphoblastic leukemia (ALL), Hodgkins disease,multiple myeloma and T-cell lymphoma. Also included are myelodysplasticsyndrome, plasma cell neoplasia, paraneoplastic syndromes, cancers ofunknown primary site as well as AIDS related malignancies.

It is to be noted that a cancer disease as well as a malignant neoplasiadoes not necessarily require the formation of metastases in distantorgans. Certain tumors exert devastating effects on the primary organitself through their aggressive growth properties. These can lead to thedestruction of the tissue and organ structure finally resulting infailure of the assigned organ function.

Compounds according to the present invention can be commerciallyapplicable for treatment, prevention or amelioration of the diseases ofbenign and malignant behavior as described before.

Neoplastic cell proliferation might effect normal cell behaviour andorgan function. For example the formation of new blood vessels, aprocess described as neovascularization, is induced by tumors or tumormetastases. Compounds according to this invention can be commerciallyapplicable for the treatment of pathophysiological relevant processescaused by benign or neoplastic cell proliferation, such as but notlimited to neovascularization by unphysiological proliferation ofvascular endothelial cells.

Drug resistance is of particular importance for the frequent failure ofstandard cancer therapeutics. This drug resistance is caused by variouscellular and molecular mechanisms like overexpression of drug effluxpumps or mutation within the cellular target protein. The commercialapplicability of compounds according to this invention is not limited to1^(st) line treatment of patents. Patients with resistance to definedcancer chemotherapeutics or target specific anti-cancer drugs (2^(nd) or3^(rd) line treatment) can be also amenable for treatment with compoundsaccording to this invention.

In the context of their properties, functions and usabilities mentionedherein, the compounds according to the present invention are expected tobe distinguished by valuable and desirable effects related therewith,such as e.g. by low toxicity, superior bioavailability in general (suchas e.g. good enteral absorption), superior therapeutic window, absenceof significant side effects, and/or further beneficial effects relatedwith their therapeutic and pharmaceutical suitability.

Due to their cellular anti-proliferative properties, compounds accordingto the present invention may be also commercially usable for treatmentof diseases associated with cell cycle and cell proliferation, such as,besides cancer discussed above, for example, fibroproliferative anddifferentiative disorders, psoriasis, rheumatoid arthritis,atherosclerosis, hyperplasia, restenosis, cardiac hypertrophy,(auto)immune disorders, fungal disorders, bone diseases, or acute orchronic inflammation.

The invention further includes a method for treating(hyper)proliferative diseases and/or disorders responsive to theinduction of apoptosis, particularly those diseases, disorders,conditions or illnesses mentioned above, in mammals, including humans,suffering therefrom comprising administering to said mammals in needthereof a pharmacologically active and therapeutically effective andtolerable amount of one or more of the compounds according to thisinvention.

The present invention further includes a method useful to modulateapoptosis and/or aberrant cell growth in the therapy of benign ormalignant neoplastic diseases, such as e.g. cancer, comprisingadministering to a subject in need of such therapy a pharmacologicallyactive and therapeutically effective and tolerable amount of one or moreof the compounds according to this invention.

The invention further includes a method for inhibiting Eg5 activity incells comprising administering a pharmacologically active andtherapeutically effective and tolerable amount of one or more of thecompounds according to this invention to a patent in need of suchinhibition.

The present invention further includes a method to modulate the mitoticspindle, i.e., for example, altering mitotic spindle formation,including decreasing spindle formation, or increasing or decreasingspindle pole separation causing malformation of the mitotic spindlepoles, comprising administering a pharmacologically active andtherapeutically effective and tolerable amount of one or more of thecompounds according to this invention to a patient in need of suchmodulation.

The present invention further includes a method to inhibit mitosis incells comprising administering a pharmacologically active andtherapeutically effective and tolerable amount of one or more of thecompounds according to this invention to a patient in need of suchinhibition.

The present invention further relates to the use of the compoundsaccording to this invention for the production of pharmaceuticalcompositions which are employed for the treatment, prophylaxis,inhibition and/or amelioration of the illnesses mentioned.

The present invention further relates to the use of the compoundsaccording to this invention for the production of pharmaceuticalcompositions which can be used in the treatment, prevention oramelioration of hyperproliferative diseases of benign or malignantbehaviour and/or disorders responsive to the induction of apoptosis in amammal, such as e.g. cancer.

The present invention further relates to the use of the compoundsaccording to this invention for the production of pharmaceuticalcompositions which can be used use in the treatment, prevention oramelioration of disorders responsive to arresting of aberrant cellgrowth and/or induction of apoptosis.

The present invention further relates to pharmaceutical compositionscomprising one or more of the compounds according to this invention anda pharmaceutically acceptable carrier or diluent.

The present invention further relates to pharmaceutical compositionsmade by combining one or more of the compounds according to thisinvention and a pharmaceutically acceptable carrier or diluent.

The present invention further relates to combinations comprising one ormore of the compounds according to this invention and pharmaceuticallyacceptable auxiliaries, excipients or vehicles, e.g. for use in thetreatment, prevention or amelioration of benign or malignant neoplasia,such as e.g. cancer.

The present invention further relates to a composition consistingessentially of a therapeutically effective and tolerable amount of oneor more compounds according to this invention together with the usualpharmaceutically acceptable vehicles, diluents and/or excipients for usein therapy, e.g. for treating, preventing or amelioratinghyperproliferative diseases, such as e.g. cancer, and/or disordersresponsive to induction of apoptosis.

The present invention further relates to compounds according to thisinvention for use in therapy, such as, for example, in the treatment,prevention or amelioration of hyperproliferative diseases of benign ormalignant behaviour and/or disorders responsive to the induction ofapoptosis, such as e.g. those diseases mentioned herein, particularlycancer.

The present invention further relates to compounds according to thisinvention having anti-proliferative and/or apoptosis inducing activity.

The present invention further relates to compounds according to thisinvention having Eg5 inhibiting properties.

The present invention further relates to pharmaceutical compositionsaccording to this invention having Eg5 inhibiting properties.

The invention further relates to the use of a pharmaceutical compositioncomprising one or more of the compounds according to this invention assole active ingredient(s) and a pharmaceutically acceptable carrier ordiluent in the manufacture of pharmaceutical products for the treatmentand/or prophylaxis of the illnesses mentioned above.

The pharmaceutical compositions according to this invention can beprepared by processes which are known per se and familiar to the personskilled in the art. As pharmaceutical compositions, the compounds of theinvention (=active compounds) are either employed as such, or preferablyin combination with suitable pharmaceutical auxiliaries and/orexcipients, e.g. in the form of tablets, coated tablets, capsules,caplets, suppositories, patches (e.g. as TTS), emulsions, suspensions,gels or solutions, the active compound content advantageously beingbetween 0.1 and 95% and where, by the appropriate choice of theauxiliaries and/or excipients, a pharmaceutical administration form(e.g. a delayed release form or an enteric form) exactly suited to theactive compound and/or to the desired onset of action can be achieved.

The person skilled in the art is familiar with auxiliaries, vehicles,excipients, diluents, carriers or adjuvants which are suitable for thedesired pharmaceutical formulations, preparations or compositions onaccount of his/her expert knowledge. In addition to solvents, gelformers, ointment bases and other active compound excipients, forexample antioxidants, dispersants, emulsifiers, preservatives,solubilizers, colorants, complexing agents or permeation promoters, canbe used.

Depending upon the particular disease, to be treated or prevented,additional therapeutic active agents, which are normally administered totreat or prevent that disease, may optionally be coadministered with thecompounds according to this invention. As used herein, additionaltherapeutic agents that are normally administered to treat or prevent aparticular disease are known as appropriate for the disease beingtreated.

For example, compounds according to this invention may be combined withone or more standard therapeutic agents used for treatment of thediseases as mentioned before.

In one particular embodiment, compounds according to this invention maybe combined with one or more art-known anti-cancer agents, such as e.g.with one or more chemotherapeutic and/or target specific anti-canceragents as described below.

Examples of known chemotherapeutic anti-cancer agents frequently usedfor combination therapy include, but not are limited to (i)alkylating/carbamylatng agents such as Cyclophosphamid (Endoxan®),Ifosfamid (Holoxan®), Thiotepa (Thiothepa Lederle®), Melphalan(Alkeran®), or chloroethylnitrosourea (BCNU); (ii) platinum derivativeslike cis-platin (Platinex® BMS), oxaliplatin or carboplatin (Cabroplat®BMS); (iii) antimitotic agents/tubulin inhibitors such as vincaalkaloids (vincristine, vinblastine, vinorelbine, vinflunine), taxanessuch as Taxol (Paclitaxel®), Taxotere (Docetaxel®) and analogs as wellas new formulations and conjugates thereof, epothilones such asEpothilone B (Patupilone®), Azaepothilone (Ixabepilone®) or ZK-EPO, afully synthetic epothilone B analog; (iv) topoisomerase inhibitors suchas anthracyclines such as Doxorubicin (Adriblastin®),epipodophyllotoxines (such as Etoposide (Etopophos®) and camptothecinanalogs such as Topotecan (Hycamtin®); (v) pyrimidine antagonists suchas 5-fluorouracil (5-FU), Capecitabine (Xeloda®),Arabinosylcytosine/Cytarabin (Alexan®) or Gemcitabine (Gemzar®); (vi)purin antagonists such as 6-mercaptopurine (Puri-Nethol®), 6-thioguanineor fludarabine (Fludara®) and finally (vii) folic acid antagonists suchas methotrexate (Farmitrexat®) and pemetrexed (Alimta®).

Examples of target specific anti-cancer drug classes used inexperimental or standard cancer therapy include but are not limited to(i) kinase inhibitors such as e.g. Glivec (Imatinib®), ZD-1839/Iressa(Gefitinib®), BAY43-9006 (Sorafenib®), SU11248 (Sutent®) orOSI-774/Tarceva (Erlotinib®); (ii) proteasome inhibitors such as PS-341(Velcade®); (iii) histone deacetylase inhibitors like SAHA, PXD101,MS275, MGCD0103, CI-994, Depsipeptide/FK228, NVP-LBH589, LAQ-824,Valproic acid (VPA) and butyrates; (iv) heat shock protein 90 inhibitorslike 17-allylaminogeldanamycin (17-AAG); (v) vascular targeting agents(VATs) like combretastatin A4 phosphate or AVE8062/AC7700 andanti-angiogenic drugs like the VEGF antibody Avastin (Bevacizumab®) orthe KDR tyrosine kinase inhibitor PTK787/ZK222584 (Vatalanib®); (vi)monoclonal antibodies such as Herceptin (Trastuzumab®), MabThera/Rituxan(Rituximab®) or C225/Erbitux (Cetuximab®) or Avastin (see above) as wellas mutants and conjugates of monoclonal antibodies and antibodyfragments; (vii) oligonucleotide based therapeutics likeG-3139/Genasense (Oblimersen®); (viii) Toll-like receptor/TLR 9 agonistslike Promune®; (ix) protease inhibitors (x) hormonal therapeutics suchas anti-estrogens (e.g. Tamoxifen), anti-androgens (e.g. Flutamide orCasodex), LHRH analogs (e.g. Leuprolide, Goserelin or Triptorelin) andaromatase inhibitors.

Other known target specific anti-cancer agents which can be used forcombination therapy include bleomycin, retinoids such as all-transretinoic acid (ATRA), DNA methyltransferase inhibitors such as the2-deoxycytidine derivative Decitabine (Docagen®), alanosine, cytokinessuch as interleukin-2 or interferons such as interferon α2 orinterferon-γ, TRAIL, DR4/5 agonistic antibodies, FasL and TNF-Ragonists.

As exemplary anti-cancer agents which can be useful in the combinationtherapy according to the present invention the following drugs may bementioned, without being restricted thereto, 5 FU, actinomycin D,ABARELIX, ABCIXIMAB, ACLARUBICIN, ADAPALENE, ALEMTUZUMAB, ALTRETAMINE,AMINOGLUTETHIMIDE, AMIPRILOSE, AMRUBICIN, ANASTROZOLE, ANCITABINE,ARTEMISININ, AZATHIOPRINE, BASILIXIMAB, BENDAMUSTINE, BEXXAR,BICALUTAMIDE, BLEOMYCIN, BROXURIDINE, BUSULFAN, CAPECITABINE,CARBOPLATIN, CARBOQUONE, CARMUSTINE, CETRORELIX, CHLORAMBUCIL,CHLORMETHINE, CISPLATIN, CLADRIBINE, CLOMIFENE, CYCLOPHOSPHAMIDE,DACARBAZINE, DACLIZUMAB, DACTINOMYCIN, DAUNORUBICIN, DESLORELIN,DEXRAZOXANE, DOCETAXEL, DOXIFLURIDINE, DOXORUBICIN, DROLOXIFENE,DROSTANOLONE, EDELFOSINE, EFLORNITHINE, EMITEFUR, EPIRUBICIN,EPITIOSTANOL, EPTAPLATIN, ERBITUX, ESTRAMUSTINE, ETOPOSIDE, EXEMESTANE,FADROZOLE, FINASTERIDE, FLOXURIDINE, FLUCYTOSINE, FLUDARABINE,FLUOROURACIL, FLUTAMIDE, FORMESTANE, FOSCARNET, FOSFESTROL, FOTEMUSTINE,FULVESTRANT, GEFITINIB, GEMCITABINE, GLIVEC, GOSERELIN, GUSPERIMUS,HERCEPTIN, IDARUBICIN, IDOXURIDINE, IFOSFAMIDE, IMATINIB, IMPROSULFAN,INFLIXIMAB, IRINOTECAN, LANREOTIDE, LETROZOLE, LEUPRORELIN, LOBAPLATIN,LOMUSTINE, MELPHALAN, MERCAPTOPURINE, METHOTREXATE, METUREDEPA,MIBOPLATIN, MIFEPRISTONE, MILTEFOSINE, MIRIMOSTIM, MITOGUAZONE,MITOLACTOL, MITOMYCIN, MITOXANTRONE, MIZORIBINE, MOTEXAFIN,NARTOGRASTIM, NEBAZUMAB, NEDAPLATIN, NILUTAMIDE, NIMUSTINE, OCTREOTIDE,ORMELOXIFENE, OXALIPLATIN, PACLITAXEL, PALIVIZUMAB, PEGASPARGASE,PEGFILGRASTIM, PENTETREOTIDE, PENTOSTATIN, PERFOSFAMIDE, PIPOSULFAN,PIRARUBICIN, PLICAMYCIN, PREDNIMUSTINE, PROCARBAZINE, PROPAGERMANIUM,PROSPIDIUM CHLORIDE, RALTITREXED, RANIMUSTINE, RANPIRNASE, RASBURICASE,RAZOXANE, RITUXIMAB, RIFAMPICIN, RITROSULFAN, ROMURTIDE, RUBOXISTAURIN,SARGRAMOSTIM, SATRAPLATIN, SIROLIMUS, SOBUZOXANE, SPIROMUSTINE,STREPTOZOCIN, TAMOXIFEN, TASONERMIN, TEGAFUR, TEMOPORFIN, TEMOZOLOMIDE,TENIPOSIDE, TESTOLACTONE, THIOTEPA, THYMALFASIN, TIAMIPRINE, TOPOTECAN,TOREMIFENE, TRASTUZUMAB, TREOSULFAN, TRIAZIQUONE, TRIMETREXATE,TRIPTORELIN, TROFOSFAMIDE, UREDEPA, VALRUBICIN, VERTEPORFIN,VINBLASTINE, VINCRISTINE, VINDESINE, VINORELBINE, VOROZOLE and ZEVALIN.

The person skilled in the art is aware on the base of his/her expertknowledge of the total daily dosage(s) and administration form(s) of theadditional therapeutic agent(s) coadministered. Said total dailydosage(s) can vary within a wide range.

In practicing the present invention, the compounds according to thisinvention may be administered in combination therapy separately,sequentially, simultaneously or chronologically staggered (such as e.g.as combined unit dosage forms, as separate unit dosage forms, asadjacent discrete unit dosage forms, as fixed or non-fixed combinations,as kit-of-parts or as admixtures) with one or more standardtherapeutics, in particular art-known anti-cancer agents, such as e.g.those mentioned above (e.g. chemotherapeutic and/or target specificanti-cancer agents).

In this context, the present invention further relates to a combinationcomprising a first active ingredient, which is at least one compoundaccording to this invention, and a second active ingredient, which is atleast one art-known anti-cancer agent, such as e.g. one or more of thosementioned herein above,

for separate, sequential, simultaneous or chronologically staggered usein therapy, such as e.g. in therapy of those diseases mentioned herein.

The term “combination” according to this invention may be present as afixed combination, a non-fixed combination or a kit-of-parts.

A “fixed combination” is defined as a combination wherein the said firstactive ingredient and the said second active ingredient are presenttogether in one unit dosage or in a single entity. One example of a“fixed combination” is a pharmaceutical composition wherein the saidfirst active ingredient and the said second active ingredient arepresent in admixture for simultaneous administration, such as in aformulation. Another example of a “fixed combination” is apharmaceutical combination wherein the said first active ingredient andthe said second active ingredient are present in one unit without beingin admixture.

A “kit-of-parts” is defined as a combination wherein the said firstactive ingredient and the said second active ingredient are present inmore than one unit. One example of a “kit-of-parts” is a combinationwherein the said first active ingredient and the said second activeingredient are present separately. The components of the kit-of-partsmay be administered separately, sequentially, simultaneously orchronologically staggered.

The present invention further relates to a pharmaceutical compositioncomprising

a first active ingredient, which is at least one compound according tothis invention, and

a second active ingredient, which is at least one art-known anti-canceragent, such as e.g. one or more of those mentioned herein above, and,optionally,

a pharmaceutically acceptable carrier or diluent,

for separate, sequential, simultaneous or chronologically staggered usein therapy.

The present invention further relates to a combination productcomprising

a.) at least one compound according to this invention formulated with apharmaceutically acceptable carrier or diluent, and

b.) at least one art-known ant-cancer agent, such as e.g. one or more ofthose mentioned herein above, formulated with a pharmaceuticallyacceptable carrier or diluent.

The present invention further relates to a kit-of-parts comprising apreparation of a first active ingredient, which is a compound accordingto this invention, and a pharmaceutically acceptable carrier or diluent;a preparation of a second active ingredient, which is an art-knownant-cancer agent, such as one of those mentioned above, and apharmaceutically acceptable carrier or diluent; for simultaneous,sequential, separate or chronologically staggered use in therapy.Optionally, said kit comprises instructions for its use in therapy, e.g.to treat hyperproliferative diseases and/or disorders responsive to theinduction of apoptosis, such as e.g. cancer.

The present invention further relates to a combined preparationcomprising at least one compound according to this invention and atleast one art-known anti-cancer agent for simultaneous, sequential orseparate administration.

In this connection, the present invention further relates tocombinations, compositions, formulations, preparations or kits accordingto the present invention having anti-proliferative and/or apoptosisinducing properties.

Also in this connection, the present invention further relates tocombinations, compositions, formulations, preparations or kits accordingto the present invention having Eg5 inhibitory activity.

In addition, the present invention further relates to a method fortreating (hyper)proliferative diseases and/or disorders responsive tothe induction of apoptosis, such as e.g. cancer, in a patient comprisingadministering a combination, composition, formulation, preparation orkit as described herein to said patient in need thereof.

In addition, the present invention further relates to a method fortreating hyperproliferative diseases of benign or malignant behaviourand/or disorders responsive to the induction of apoptosis, such as e.g.cancer, in a patient comprising administering in combination therapyseparately, simultaneously, sequentially or chronologically staggered apharmaceutically active and therapeutically effective and tolerableamount of a pharmaceutical composition, which comprises a compoundaccording to this invention and a pharmaceutically acceptable carrier ordiluent, and a pharmaceutically active and therapeutically effective andtolerable amount of one or more art-known anti-cancer agents, such ase.g. one or more of those mentioned herein, to said patient in needthereof.

In further addition, the present invention relates to a method fortreating, preventing or ameliorating hyperproliferative diseases and/ordisorders responsive to induction of apoptosis, such as e.g. benign ormalignant neoplasia, particularly any of those cancer diseases mentionedherein, in a patient comprising administering a combination according tothe present invention.

In addition, the present invention further relates to the use of acomposition, combination, formulation, preparation or kit in themanufacture of a pharmaceutical product, such as e.g. a commercialpackage or a medicament, for treating, preventing or amelioratinghyperproliferative diseases, such as e.g. cancer, and/or disordersresponsive to the induction of apoptosis, particularly those diseasesmentioned herein.

The present invention further relates to the use of one or more of thecompounds according to this invention for the manufacture of amedicament for use in combination with one or more anti-cancer agents,e.g. one or more anti-cancer agents selected from chemotherapeutic andtarget-specific anti-cancer agents, such as e.g. from those mentionedherein, for the treatment of cancer, particularly for the treatment ofone of those cancer diseases mentioned above.

The present invention further relates to a commercial package comprisingone or more compounds of the present invention together withinstructions for simultaneous, sequential or separate use with one ormore chemotherapeutic and/or target specific anti-cancer agents, such ase.g. any of those mentioned herein.

The present invention further relates to a commercial package consistingessentially of one or more compounds of the present invention as soleactive ingredient together with instructions for simultaneous,sequential or separate use with one or more chemotherapeutic and/ortarget specific anti-cancer agents, such as e.g. any of those mentionedherein.

The present invention further relates to a commercial package comprisingone or more chemotherapeutic and/or target specific anti-cancer agents,such as e.g. any of those mentioned herein, together with instructionsfor simultaneous, sequential or separate use with one or more compoundsaccording to the present invention.

The compositions, combinations, preparations, formulations, kits orpackages mentioned in the context of the combination therapy accordingto this invention may also include more than one of the compoundsaccording to this invention and/or more than one of the art-knownanti-cancer agents mentioned.

The first and second active ingredient of a combination or kit-of-partsaccording to this invention may be provided as separate formulations(i.e. independently of one another), which are subsequently broughttogether for simultaneous, sequential, separate or chronologicallystaggered use in combination therapy; or packaged and presented togetheras separate components of a combination pack for simultaneous,sequential, separate or chronologically staggered use in combinationtherapy.

The type of pharmaceutical formulation of the first and second activeingredient of a combination or kit-of-parts according to this inventioncan be similar, i.e. both ingredients are formulated in separate tabletsor capsules, or can be different, i.e. suited for differentadministration forms, such as e.g. one active ingredient is formulatedas tablet or capsule and the other is formulated for e.g. intravenousadministration.

The amounts of the first and second active ingredients of thecombinations, compositions or kits according to this invention maytogether comprise a therapeutically effective amount for the treatment,prophylaxis or amelioration of a (hyper)proliferative disease and/ordisorders responsive to induction of apoptosis, such as e.g. benign ormalignant neoplasia, particularly one of those cancer diseases mentionedherein.

In addition, compounds according to the present invention can be used inthe pre- or post-surgical treatment of cancer.

In further addition, compounds of the present invention can be used incombination with radiation therapy.

A combination according to this invention can refer to a compositioncomprising both the compound(s) according to this invention and theother active anti-cancer agent(s) in a fixed combination (fixed unitdosage form), or a medicament pack comprising the two or more activeingredients as discrete separate dosage forms (non-fixed combination).In case of a medicament pack comprising the two or more activeingredients, the active ingredients are preferably packed into blistercards which are suited for improving compliance.

Each blister card preferably contains the medicaments to be taken on oneday of treatment. If the medicaments are to be taken at different timesof day, the medicaments can be disposed in different sections on theblister card according to the different ranges of times of day at whichthe medicaments are to be taken (for example morning and evening ormorning, midday and evening). The blister cavities for the medicamentsto be taken together at a particular time of day are accommodated in therespective range of times of day. The various times of day are, ofcourse, also put on the blister in a clearly visible way. It is alsopossible, of course, for example to indicate a period in which themedicaments are to be taken, for example stating the times.

The daily sections may represent one line of the blister card, and thetimes of day are then identified in chronological sequence in thiscolumn.

Medicaments which must be taken together at a particular time of day areplaced together at the appropriate time on the blister card, preferablya narrow distance apart, allowing them to be pushed out of the blistereasily, and having the effect that removal of the dosage form from theblister is not forgotten.

The administration of the pharmaceutical compositions or combinationsaccording to the invention may be performed in any of the generallyaccepted modes of administration available in the art. Illustrativeexamples of suitable modes of administration include intravenous, oral,nasal, parenteral, topical, transdermal and rectal delivery. Oral andintravenous delivery are preferred.

For the treatment of dermatoses, the compounds of the invention can bein particular administered in the form of those pharmaceuticalcompositions which are suitable for topical application. For theproduction of the pharmaceutical compositions, the compounds of theinvention (=active compounds) are preferably mixed with suitablepharmaceutical auxiliaries and further processed to give suitablepharmaceutical formulations. Suitable pharmaceutical formulations are,for example, powders, emulsions, suspensions, sprays, oils, ointments,fatty ointments, creams, pastes, gels or solutions.

The pharmaceutical compositions according to the invention can beprepared by processes known per se. The dosage of the active compoundsis carried out in the order of magnitude customary for Eg5 inhibitors,inhibitors for cellular proliferation or apoptosis inducers. Topicalapplication forms (such as ointments) for the treatment of dermatosesthus contain the active compounds in a concentration of, for example,0.1-99%. The customary dose in the case of systemic therapy (p.o.) isbetween 0.03 and 30 mg/kg per day, (i.v.) is between 0.03 and 30mg/kg/h. In another embodiment, the dose in the case of systemic therapy(p.o.) is between 0.3 and 30 mg/kg per day, (i.v.) is between 0.3 and 30mg/kg/h.

Biological Investigations

The ATPase activity of Eg5 kinesin motor domains (Cytoskeleton, cat. No.EG01) can be used to monitor the effects of modulating agents. The testcompounds are dissolved as 10 mM solutions in dimethylsulfoxide (DMSO).1 μl of appropriate DMSO dilutions of the test compounds are added toeach well of a 96 well flat bottom plate. Each compound dilution istested as duplicates. The reagents are added and the final reaction ofthe standard assay contains 15 mM Pipes, pH 6.8, 5.0 mM MgCl₂, 0.5 mMKCl, 1 mM EGTA, 0.1 mg/ml BSA, 0.025% Tween 20, 2 mM Glutathion, 5 μMPaclitaxel, 200 nM preformed microtubules (Cytoskeleton, cat. No.MT001), 300 μM ATP, and Eg5 protein (50 ng) in a reaction volume of 100μl. The controls include buffer wells with ATP and 1% DMSO. Reactionsare started by the addition of ATP, incubated at room temperature for 30min., and terminated by removing 20 μl of the reaction volume and addingit to 80 μl of 1 M perchloric acid, followed by the addition of 80 μlMalachite green reagent. Malachite green reagent is prepared by mixing asolution of 4.2 g ammonium molybdate in 100 ml 4 N HCl with a solutionof 0.135 g Malachite green in 300 ml H₂O. The reactions are incubatedfor a further 20 min. and then read at 615 nm.

The corresponding IC₅₀ values of the compounds for Eg5 inhibition aredetermined from the concentration-effect curves.

Representative inhibitory values [measured as −log IC₅₀ (mol/l)]determined in the aforementioned assay follow from the following tableA, in which the numbers of the compounds correspond to the numbers ofthe examples.

TABLE A Inhibition of Eg5 activity Compound −log IC₅₀ [mol/l] 1, 2, 3, 4and 5 The inhibitory values of these listed compounds are all ≧6.9

The anti-proliferative/cytotoxic activity of the compounds describedherein can be tested on subclones of RKO human colon adenocarcinomacells (Schmidt et al., Oncogene 19, 2423-2429; 2000) using the AlamarBlue cell viability assay (described in O'Brien et al. Eur J Biochem267, 5421-5426, 2000). The compounds are dissolved as 10 mM solutions inDMSO and subsequently diluted in semi-logarithmic steps. DMSO dilutionsare further diluted 1:100 into Dulbecco's modified Eagle's medium (DMEM)containing 10% fetal calf serum to a final concentration twice as muchas the final concentration in the test. RKO subclones are seeded into 96well flat bottom plates at a density of 4000 cells per well in a volumeof 50 μl per well. 24 hours after seeding the 50 μl each of the compounddilutions in DMEM medium are added into each well of the 96 well plate.Each compound dilution is tested as quadruplicates. Wells containinguntreated control cells are filled with 50 μl DMEM medium containing 1%DMSO. The cells are then incubated with the substances for 72 hours at37° C. in a humidified atmosphere containing 5% carbon dioxide. Todetermine the viability of the cells, 10 μl of an Alamar Blue solution(Biosource) are added and the fluorescence is measured at an extinctionof 544 nm and an emission of 590 nm. For the calculation of the cellviability the emission value from untreated cells is set as 100%viability and the emission rates of treated cells are set in relation tothe values of untreated cells. Viabilities are expressed as % values.

The corresponding IC₅₀ values of the compounds foranti-proliferative/cytotoxic activity are determined from theconcentration-effect curves.

To determine the cell cycle specific mode of action, subclones of RKOcolon adenocarcinoma cells (RKOp27 as described by Schmidt et al. inOncogene 19, 2423-2429; 2000) are seeded into 96 well flat bottom platesat a density of 16000 cells per well in a volume of 50 μl per well inDMEM growth medium with 10% FCS containing 10 μM Ponasterone A. 24 hoursafter seeding the 50 μl each of the compound dilutions in DMEM mediumare added into each well of the 96-well plate. Each compound dilution istested as quadruplicates. Wells containing untreated control cells arefilled with 50 μl DMEM medium containing 1% DMSO. The cells are thenincubated with the substances for 72 hours at 37° C. in a humidifiedatmosphere containing 5% carbon dioxide. To determine the viability ofthe cells, 10 μl of an Alamar Blue solution (Biosource) are added andthe fluorescence is measured at an extinction of 544 nm and an emissionof 590 nm. For the calculation of the cell viability the emission valuefrom untreated cells is set as 100% viability and the emission rates oftreated cells are set in relation to the values of untreated cells.Viabilities are expressed as % values. Viability is compared ofproliferating cells grown in the absence of the inducer Ponasterone A,versus viability of cells arrested by the expression of ectopic p27Kip1induced by Ponasterone A.

Representative values for anti-proliferation/cytotoxicity [measured as−log IC₅₀ (mol/l)] determined in the aforementioned assays follow fromthe following table B, in which the numbers of the compounds correspondto the numbers of the examples.

TABLE B Anti-proliferative/cytotoxic activity −log IC₅₀ [mol/l] −logIC₅₀ [mol/l] Compound RKO p27 uninduced RKO p27 induced 1 The −logIC₅₀values of The −logIC₅₀ values of these 2 these listed compounds listedcompounds are all ≦4 3 are all ≧6.2

The induction of apoptosis can be measured by using a Cell deathdetection ELISA (Roche Biochemicals, Mannheim, Germany). RKOp27 coloncancer cells are seeded into 96 well flat bottom plates at a density of10000 cells per well in a volume of 50 μl RPMI medium (containing 10%fetal calf serum) per well. 24 hours after seeding the 50 μl each of thecompound dilutions in RPMI medium are added into each well of the 96Well plate. Each compound dilution is tested at least as triplicates.Wells containing untreated control cells are filled with 50 μl RPMImedium containing 1% DMSO. The cells are then incubated with thesubstances for 24 hours at 37° C. in a humidified atmosphere containing5% carbon dioxide. As a positive control for the induction of apoptosis,cells are treated with 50 μM Cisplatin (Gry Pharmaceuticals,Kirchzarten, Germany). Medium is then removed and the cells are lysed in200 μl lysis buffer. After centrifugation as described by themanufacturer, 10 μl of cell lysate is processed as described in theprotocol. The degree of apoptosis is calculated as follows: Theabsorbance at 405 nm obtained with lysates from cells treated with 50 μMcisplatin is set as 100 cpu (cisplatin units), while an absorbance at405 nm of 0.0 was set as 0.0 cpu. The degree of apoptosis is expressedas cpu in relation to the value of 100 cpu reached with the lysatesobtained from cells treated with 50 μM cisplatin. The corresponding EC₅₀values of the compounds for apoptosis inducing activity are determinedfrom the concentration-effect curves.

Representative values for induction of apoptosis [measured as −log EC₅₀(mol/l)] determined in the aforementioned assays follow from thefollowing table C, in which the numbers of the compounds correspond tothe numbers of the examples.

TABLE C Apoptosis Induction Compound −log EC₅₀ [mol/l] 1, 2, 3 and 5 The−logEC₅₀ values of these listed compounds are all ≧5.6

Experimental perturbation of Eg5 function causes a characteristicmalformation of the mitotic spindle, which can be examined by confocallaser scanning microscopy. NCI-H460 non-small cell lung cancer cells aregrown overnight on glass cover slips (Nunc™ Lab-Tek™ Chamber Slides) in1800 μl DMEM medium containing 10% fetal calf serum. The test compoundsare dissolved as 10 mM solutions in DMSO. Appropriate DMSO dilutions ofthe test compounds are further diluted 1:10 into DMEM medium containing10% fetal calf serum to a final concentration ten times as much as thefinal concentration in the test. 24 hours after seeding, 200 μl of thecompound dilutions in DMEM medium are added into each well of the coverslip. As a control, 200 μl DMEM medium containing 10% DMSO are added. 24hours after incubation with the test compounds, the cells are washedwith PBS, and fixed with 3.7% formaldehyde in H₂O for 20 min. at 37° C.Subsequently, cells are washed with PBS and incubated with 0.1% TritonX-100 in a buffer containing 1.471 mM KH₂PO₄, 8.504 mM Na₂HPO₄, 137 mMNaCl, 1.325 mM CaCl₂, 2.685 mM KCl, 0.542 mM MgCl₂, pH 7.2 for 15 min.at room temperature. For saturation of non-specific binding, cells areincubated in 2% BSA/10% FCS in PBS (=blocking buffer) for 30 min. atroom temperature prior to incubation with anti-alpha tubulin monoclonalantibodies (Sigma, #T5168; 1:1000), followed by Cy3-conjugated rabbitanti-mouse IgG (H+L) antibody (Jackson Immuno Research; 1:1000). Allantibody incubations are performed for one hour at 37° C. in blockingbuffer, and cells are washed three times in PBS between differentincubations. DNA is counterstained with Hoechst 33342 (0.1 μg/ml).Coverslips are mounted in Vectashield (Vector Laboratories, Burlingame,Calif.) and examined with a Leica TCS SP2 confocal laser scanningmicroscope fitted with appropriate filters (Leica Microsystems,Bensheim, Germany). Representatively, the compounds numbered as Example1 and Example 3 in this invention are tested in the aforementioned assay(application of each 1 μM) and are found to produce a significantenrichment of cells arrested in mitosis with rosettes of condensedmitotic chromosomes attached to radial arrays of microtubules.

1. A compound of formula (I*)

in which R1 is 1-4C-alkyl, 3-7C-cycloalkyl, 3-7C-cycloalkyl-1-4C-alkyl,or 2-7C-alkyl substituted by R11, R11 is —N(R111)R112, R111 is hydrogen,or 1-4C-alkyl, R112 is hydrogen, or 1-4C-alkyl, or R111 and R112together, and with inclusion of the nitrogen atom to which they arebonded, form a ring Het, Het is piperidinyl, morpholinyl,thiomorpholinyl, or pyrrolidinyl, R2 is hydrogen, 1-4C-alkyl, halogen,trifluoromethyl, 1-4C-alkoxy, hydroxyl, 1-4C-alkoxy-2-4C-alkoxy,hydroxy-2-4C-alkoxy, 3-7C-cycloalkoxy, 3-7C-cycloalkyl-1-4C-alkoxy, orcompletely or predominantly fluorine-substituted 1-4C-alkoxy, R3 ishydrogen, or 1-4C-alkoxy, and X is NH, oxygen or sulphur, and R4 is1-4C-alkyl, or X is sulphur, and R4 is hydrogen, or X is oxygen, and R4is hydrogen, or a salt, stereoisomer or a salt of a stereoisomerthereof.
 2. A compound according to claim 1, in which R1 is 1-2C-alkyl,cyclopropyl, cyclopropylmethyl, or 2-4C-alkyl substituted by R11, R11 is—N(R111)R112, R111 is 1-2C-alkyl, R112 is 1-2C-alkyl, or R111 and R112together, and with inclusion of the nitrogen atom to which they arebonded, form a ring Het, Het is piperidinyl, morpholinyl,thiomorpholinyl, or pyrrolidinyl, R2 is hydrogen, R3 is hydrogen, and Xis NH, oxygen or sulphur, and R4 is methyl, or X is sulphur, and R4 ishydrogen, or X is oxygen, and R4 is hydrogen, or a salt, stereoisomer ora salt of a stereoisomer thereof.
 3. A compound according to claim 1, inwhich R1 is 1-4C-alkyl, 3-7C-cycloalkyl, 3-7C-cycloalkyl-1-4C-alkyl, or2-7C-alkyl substituted by R11, R11 is —N(R111)R112, R111 is hydrogen, or1-4C-alkyl, R112 is hydrogen, or 1-4C-alkyl, or R111 and R112 together,and with inclusion of the nitrogen atom to which they are bonded, form aring Het, Het is piperidinyl, morpholinyl, thiomorpholinyl, orpyrrolidinyl, R2 is hydrogen, 1-4C-alkyl, halogen, trifluoromethyl,1-4C-alkoxy, hydroxyl, 1-4C-alkoxy-2-4C-alkoxy, hydroxy-2-4C-alkoxy,3-7C-cycloalkoxy, 3-7C-cycloalkyl-1-4C-alkoxy, or completely orpredominantly fluorine-substituted 1-4C-alkoxy, R3 is hydrogen, or1-4C-alkoxy, X is NH, oxygen or sulphur, and R4 is 1-4C-alkyl, or asalt, stereoisomer or a salt of a stereoisomer thereof.
 4. A compoundaccording to claim 1, in which R1 is 1-4C-alkyl, 3-7C-cycloalkyl,3-7C-cycloalkyl-1-4C-alkyl, or 2-7C-alkyl substituted by R11, R11 is—N(R111)R112, R111 is hydrogen, or 1-4C-alkyl, R112 is hydrogen, or1-4C-alkyl, or R111 and R112 together, and with inclusion of thenitrogen atom to which they are bonded, form a ring Het, Het ispiperidinyl, morpholinyl, thiomorpholinyl, or pyrrolidinyl, R2 ishydrogen, 1-4C-alkyl, halogen, trifluoromethyl, 1-4C-alkoxy, hydroxyl,1-4C-alkoxy-2-4C-alkoxy, hydroxy-2-4C-alkoxy, 3-7C-cycloalkoxy,3-7C-cycloalkyl-1-4C-alkoxy, or completely or predominantlyfluorine-substituted 1-4C-alkoxy, R3 is hydrogen, or 1-4C-alkoxy, X issulphur, and R4 is hydrogen, or a salt, stereoisomer or a salt of astereoisomer thereof.
 5. A compound according to claim 1, in which R1 ismethyl, ethyl, or cyclopropyl, R2 is hydrogen, R3 is hydrogen, X is NH,oxygen or sulphur, and R4 is methyl, or a salt, stereoisomer or a saltof a stereoisomer thereof.
 6. A compound according to claim 1, in whichR1 is methyl, ethyl, or cyclopropyl, R2 is hydrogen, R3 is hydrogen, Xis sulphur, and R4 is hydrogen, or a salt, stereoisomer or a salt of astereoisomer thereof.
 7. A compound according to claim 1, in which R1 ismethyl, ethyl, or ethyl substituted by R11, R11 is —N(R111)R112, R111 ismethyl, R112 is methyl, or R111 and R112 together, and with inclusion ofthe nitrogen atom to which they are bonded, form a ring Het, Het ismorpholinyl, R2 is hydrogen, R3 is hydrogen, and X is NH, oxygen orsulphur, and R4 is methyl, or X is sulphur, and R4 is hydrogen, or X isoxygen, and R4 is hydrogen, or a pharmaceutically acceptable saltthereof.
 8. A compound according to claim 1, in which R1 is methyl, orethyl substituted by R11, R11 is —N(R111)R112, R111 is methyl, R112 ismethyl, R2 is hydrogen, R3 is hydrogen, and X is NH, and R4 is methyl,or X is sulphur, and R4 is methyl, or X is oxygen, and R4 is methyl, ora pharmaceutically acceptable salt thereof.
 9. A compound according toclaim 1, in which R1 is methyl, or ethyl substituted by R11, R11 is—N(R111)R112, R111 is methyl, R112 is methyl, R2 is hydrogen, R3 ishydrogen, and X is sulphur, and R4 is hydrogen, or X is oxygen, and R4is hydrogen, or a pharmaceutically acceptable salt thereof.
 10. Acompound according to claim 1, in which R1 is methyl, R2 is hydrogen, R3is hydrogen, and X is NH, and R4 is methyl, or X is sulphur, and R4 ismethyl, or X is oxygen, and R4 is methyl, or a pharmaceuticallyacceptable salt thereof.
 11. A compound according to claim 1, in whichR1 is methyl, R2 is hydrogen, R3 is hydrogen, and X is sulphur, and R4is hydrogen, or X is oxygen, and R4 is hydrogen, or a pharmaceuticallyacceptable salt thereof.
 12. A compound according to claim 1, in whichR2 and R3 are both hydrogen, and R1, R4 and X have any one of themeanings 1.1 to 1.6 given in the following table: R1 R4 X 1.1 —CH₃ —CH₃NH 1.2 —CH₃ —CH₃ S 1.3 —CH₂CH₂—N(CH₃)₂ —CH₃ NH 1.4 —CH₂CH₂—N(CH₃)₂ —CH₃S 1.5 —CH₃ H S 1.6 —CH₂CH₂—N(CH₃)₂ H S

or a pharmaceutically acceptable salt thereof.
 13. A pharmaceuticalcomposition comprising a therapeutically effective amount of one or morecompounds according to claim 1, or a salt, stereoisomer or a salt of astereoisomer thereof, together with a pharmaceutically acceptableexcipient and/or vehicle.
 14. A compound of according to claim 1, inwhich R4 is 2-4C-alkyl.
 15. A compound of according to claim 1, in whichR1 is 3-7C-cycloalkyl.
 16. A compound of according to claim 1, in whichR1 is 3-7C-cycloalkyl-1-4C-alkyl.
 17. A compound of according to claim1, in which R1 is 2-7C-alkyl substituted by R11.
 18. A compoundaccording to claim 1, selected from the group consisting of(3aS,10R)-10-(3-Hydroxy-phenyl)-2,3a-dimethyl-3a,4,9,10-tetrahydro-2,9,10a-triaza-cyclopenty[b]fluorene-1,3-dione;(3aS,10R)-10-(3-Hydroxy-phenyl)-2-methyl-4,10-dihydro-3aH-9-thia-2,10-diaza-cyclopenta[b]fluorene-1,3-dione;(3aS,10R)-2-Butyl-10-(3-hydroxy-phenyl)-4,10-dihydro-3aH-9-thia-2,10a-diaza-cyclopenta[b]fluorene-1,3-dione;(3aS,10R)-10-(3-Hydroxy-phenyl)-2,3a-dimethyl-4,10-dihydro-3aH-9-thia-2,10a-diaza-cyclopenta[b]fluorene-1,3-dione;(3aS,10R)-2-(2-Dimethylamino-ethyl)-10-(3-hydroxy-phenyl)-3a,4,9,10-tetrahydro-2,9,10a-triaza-cyclopenty[b]fluorene-1,3-dione;(3aS,10R)-2-(2-Dimethylamino-ethyl)-10-(3-hydroxy-phenyl)-4,10-dihydro-3aH-9-thia-2,10-diaza-cyclopenta[b]fluorene-1,3-dione;(3aS,10R)-2-(2-Dimethylamino-ethyl)-10-(3-hydroxy-phenyl)-3a-methyl-4,10-dihydro-3aH-9-thia-2,10a-diaza-cyclopenta[b]fluorene-1,3-dione.19. A compound selected from the group consisting of(±)-(3aSR,10RS)-10-(3-Hydroxy-phenyl)-2,3a-dimethyl-3a,4,9,10-tetrahydro-2,9,10a-triaza-cyclopenty[b]fluorene-1,3-dione;(3aSR,10RS)-10-(3-Hydroxy-phenyl)-2,3a-dimethyl-4,10-dihydro-3aH-9-thia-2,10a-diaza-cyclopenta[b]fluorene-1,3-dione;(3aSR,10RS)-2-(2-Dimethylamino-ethyl)-10-(3-hydroxy-phenyl)-3a-methyl-3a,4,9,10-tetrahydro-2,9,10a-triaza-cyclopenty[b]fluorene-1,3-dione;and(3aSR,10RS)-2-(2-Dimethylamino-ethyl)-10-(3-hydroxy-phenyl)-3a-methyl-4,10-dihydro-3aH-9-thia-2,10a-diaza-cyclopenta[b]fluorene-1,3-dione.20. A compound according to claim 2, in which R1 is 2-4C-alkylsubstituted by R11, R11 is —N(R111)R112, R111 is 1-2C-alkyl, R112 is1-2C-alkyl, R2 is hydrogen, R3 is hydrogen, X is NH, R4 is methyl, or apharmaceutically acceptable salt thereof.
 21. A compound according toclaim 3, in which R1 is 2-7C-alkyl substituted by R11, R11 is—N(R111)R112, R111 is hydrogen, or 1-4C-alkyl, R112 is hydrogen, or1-4C-alkyl, R2 is hydrogen, 1-4C-alkyl, halogen, trifluoromethyl,1-4C-alkoxy, hydroxyl, 1-4C-alkoxy-2-4C-alkoxy, hydroxy-2-4C-alkoxy,3-7C-cycloalkoxy, 3-7C-cycloalkyl-1-4C-alkoxy, or completely orpredominantly fluorine-substituted 1-4C-alkoxy, R3 is hydrogen, or1-4C-alkoxy, X is NH, and R4 is 1-4C-alkyl, or a pharmaceuticallyacceptable salt thereof.
 22. A compound according to claim 21, in whichR112 is hydrogen, R2 is hydrogen, and R3 is hydrogen, or apharmaceutically acceptable salt thereof.
 23. A compound according toclaim 1, in which X is NH, and R4 is methyl.
 24. A compound according toclaim 23, in which R2 and R3 are both hydrogen.
 25. A compound accordingto claim 24, in which R1 is 2-(N,N-dimethylamino)-ethyl.
 26. A compoundaccording to claim 24, in which R1 is 2-4C-alkyl substituted by R11, R11is —N(R111)R112, R111 is hydrogen, or 1-4C-alkyl, R112 is hydrogen, or1-4C-alkyl.
 27. A compound according to claim 26, in which R111 ishydrogen, and R112 is 3-4C-alkyl.
 28. A method for treating colon cancercomprising administering to a subject in need thereof an effectiveamount of a compounds of claim 1.